We have demonstrated that female rats experience significantly decreased brain injury after experimental stroke compared to male rats. Ovariectomy eliminates the protection from stroke injury enjoyed by female rats, suggesting that female reproductive hormones are neuroprotective. Furthermore, we have shown that exogenous estrogen is neuroprotective in multiple models of stroke. Two estrogen receptors, ER-alpha and ER-beta, act in neurons via a number of mechanisms. Estrogen receptors directly regulate gene transcription and, in addition, trigger the MAP kinase signaling cascade. The present proposal focuses on the molecular mechanism of how estrogen protects the brain from stroke. The overall hypothesize of this proposal is that estrogen protects the brain during stroke by acting upon estrogen receptors within neurons of the brain. We will use a tissue culture model of neuronal injury to address several specific questions regarding estrogen's action on neurons. In Aim 1, we will determine whether estrogen receptors are necessary for estrogen-mediated protection by examining properties of neurons in which receptors have been inactivated. In Aim 2, we will evaluate whether activation of estrogen receptor-mediated transcription is sufficient for neuroprotection by utilizing molecular mutants of ER which constitutively activate estrogen signaling. In Aim 3, we will determine whether Src/Ras/MAP kinase activation, known to occur during estrogen stimulation of neurons, plays a role in protection. In Aim 4, we will ask whether our in vitro mechanism is valid in the intact animal using viral mediated gene transfer into an in vivo model of stroke. The mechanism of how estrogen affects stroke will perhaps foster development of novel treatments for stroke, and may influence hormone replacement regimens for postmenopausal women.